This invention relates to molecular biology, and in particular, to the use of an applied electric field in a microfluidic system for the manipulation of biological samples comprising cells and cell lysate(s) for subsequent analysis.
Interest in microfabricated devices for chemical sensing and analysis has grown substantially over the past decade, primarily because these miniature devices have the potential to provide information rapidly and reliably at low cost. Microchips fabricated on planar substrates are advantageous for manipulating small sample volumes, rapidly processing materials, and integrating sample pretreatment and separation strategies. The ease with which materials can be manipulated and the ability to fabricate structures with interconnecting channels that have essentially no dead volume contribute to the high performance of these devices. See, for example U.S. Pat. Nos. 5,858,195 and 6,001,229, which are commonly owned with this application. To carry out a complete analysis, many different kinds of functional elements can be designed and integrated on microchips. These elements include filters, valves, pumps, mixers, reactors, separators, cytometers and detectors, which can be operatively coupled together under computer control to enable the implementation of a wide range of microchip-based analyses.
One area of particular interest is the analysis of cells and cell populations. At present most techniques for cellular analysis depend upon pooling a population of cells to obtain a large enough quantity of analyte for detection. Pooling of cells, however, obscures any variation in analyte concentration from cell to cell. For many studies average analyte values across a large population of cells will be acceptable; however, for the study of processes such as carcinogenesis, the ability to quantitate analytes in individual cells is required so that rare mutations in cells, which lead to drastic changes in cell metabolism and progression to cancer, can be detected.
With the advances in Capillary Electrophoresis over the last two decades the quantitation of analytes in individual cells has become feasible albeit slow because of the intensive manual manipulations which have to be performed. The potential to automate and integrate cell transport, manipulation and lysis with separation and analyte detection make microfluidic devices a desirable platform for performing high throughput screening of individual cells from large populations. A key step in integrating cell handling with analyte detection and quantitation is providing a method of cell lysis which is rapid and generates small axial extent plugs for subsequent analysis. Because the resolution or separation between any pair of compounds can be detrimentally affected by long injection plug lengths, small axial extent plugs are important for a successful separation. It should be noted, however, that while small axial extent plugs are desired, the contents of the cell after lysis should be spread over several cell volumes so that reaction pathways within the cell are terminated and proteolytic enzymes released from vesicles during lysis are sufficiently diluted. This will prevent the possible digestion of proteins of interest.
The use of an applied electric field for cell lysis is known. For example, the lysis of erythrocytes in suspension by pulsed electric fields has been reported both for bovine (Sale and Hamilton, xe2x80x9cEffects of High Electric Fields on Microorganisms III, Lysis of Erythrocytes and Protoplastsxe2x80x9d, Biochim et Biophys Acta, 163:37 (1967)) and human erythrocytes (Kinosita and Tsong, xe2x80x9cVoltage-Induced Pore Formation and Hemolysis of Human Erythrocytesxe2x80x9d, Biochim et Biophys Acta, 471:227 (1977); and Kinosita and Tsong, xe2x80x9cHemolysis of Human Erythrocytes by a Transient Electric Filedxe2x80x9d, Proc Nail Acad Sci., 74:1923 (1977)). These reports indicate that applied electric fields resulting in cellular transmembrane potentials on the order of 1 Volt can result in lysis of erythrocytes. However, these previously reported cell lysis techniques utilizing an electric field are typically carried out in a macroscale device, rather than a microchip device. Consequently, such techniques are lacking in certain respects. Specifically, the conditions employed in macroscale electric cell lysis devices do not consistently release relatively high molecular weight nucleic acid molecules, because such molecules do not readily pass through the pores created in the cell membrane by this lysis technique. Also, the existing macroscale, electric lysis devices function as stand-alone units, thus precluding integration of cell manipulation and lysis with separation and analysis of cell lysate in a unitary device.
Other methods of cell manipulation and/or lysis on microscale devices have been proposed. See, for example, U.S. Pat. Nos. 4,676,274 and 5,304,487. Chemical lysis on a microchip device has been demonstrated by mixing a surfactant, i.e. sodium dodecylsulfate, with canine erythrocyte cells. (Li and Harrison, Anal. Chem., 69:1564-1568 (1997)). This report indicates that the cells were lysed in under 0.3 sec. No subsequent analysis of the cellular contents, however, was reported. Single erythrocytes have been lysed and the cell contents separated using two capillaries across which an electric field is applied. A gap of about 5 xcexcm is provided between the two capillaries. As intact cells pass through the gap, they are lysed and the contents of the lysed cells are transported to the second capillary for separation. The lysis is presumed to be caused by the mechanical shear stresses induced by the change in electric field strength between the capillaries and the gap region. The gap region is considerably larger in cross sectional area, so that the field strength and, therefore, cell velocity is lower than in the capillaries. See, Chen and Lillard, Anal. Chem., 73:111-18 (2001).
Cell lysis can be considered an extreme form of cell membrane permeablization (poration). Optoporation has been carried out using highly focused light from a pulsed laser (Soughhayer et al., Anal. Chem. 72(6): 1342-1347 (2000)). The laser is focused near the cell at the aqueous/glass interface. When the laser is pulsed, a stress (shock) wave is generated which transiently permeablizes the cell. This technique has been shown to be capable of lysing cells, as well. The occurrence of cell lysis or poration only is a function of the cell""s distance from the laser focal spot.
Although the above-mentioned cell lysis techniques of the prior art are useful for certain applications, there exists a need in the art for microchip-based cell manipulations and lysis which is sufficiently rapid to minimize continued cellular activity after lysis, thus producing greater accuracy in the analysis of cell processes.
In accordance with one aspect of the present invention, there is provided a method of releasing the intracellular contents of at least one cell of a cell-containing fluid sample for analysis. The method of the invention comprises providing a substrate having a microchannel structure which includes one or more microchannel(s). An electric field is generated from a source of electric potential and applied in a spatially defined region of the aforementioned microchannel, which functions as a cell lysis region. The strength of the applied electric field is adequate to induce cell lysis. At least one cell of the fluid sample is positioned in the cell lysis region for a time sufficient to release the intracellular contents into the fluid sample. The released intracellular contents form an analyte plug of narrow axial extent in the microchannel.
According to another aspect of the invention, there is provided a microfluidic system for transport and lysis of at least one cell of a cell-containing fluid sample. The microfluidic system of this embodiment of the invention comprises a source of electric potential and a solid substrate including one or more microchannel(s) with a longitudinal axis, and a cell lysis region between first and second electrical contacts positioned adjacent (i.e. on or in close proximity to) microchannel wall portions on different sides of the longitudinal axis. The electrical contacts, which are spatially separated by the cell lysis region and electrically isolated from one another, are connected to a source of electric potential, which is operative to apply an electric field to the cell lysis region, transverse to the fluid sample flow path, within the microchannel space between the electrical contacts. The system also includes means for transporting the cell-containing fluid sample along the aforementioned microchannel.
According to a further aspect of this invention, there is provided a microfluidic system for transport and lysis of at least one cell of a cell-containing fluid sample and separation of its intracellular content. This system comprises a solid substrate having one or more microchannel(s) disposed therein, which includes a cell transport segment and a separation segment with first and second end portions. A first and a second electrode are provided along the aforementioned microchannel, intermediate the transport segment and the separation segment, and are spatially separated from one another, defining a space in the microchannel between them that serves as a cell lysis region. The electrodes are connected to a source of electric potential to apply an electric field to the cell lysis region. This system further includes means for flowing the cell-containing fluid sample through the aforementioned microchannel and means for applying an electric potential difference between the first and second separation segment end portions for effecting separation of the intracellular contents of lysed cells.
The microfluidic system and methods of this invention enable examination of the contents of single cells with high throughput. Consequently, this invention is expected to have considerable utility for facilitating research in the life sciences, and especially the pharmaceutical industry. For example, implementation of this invention in the pharmaceutical industry could expedite screening of cellular responses to large combinatorial libraries of potential novel drugs. In addition, the present invention can be used to advantage for the study of carcinogenesis or oncogenesis by assisting in the detection of rare cell mutations at an early stage, which is considered essential to the successful treatment of various forms of cancer. This invention may also be used to further elucidate metabolic pathways in cells.